1. Field of the Invention
The field of the invention is measuring or detecting processes involving cells, nucleic acids, or proteins, including enzymes, devices therefor, and processes for forming such devices.
2. Description of Related Art
Current methods used for detection and quantification of protein biomarkers include techniques like the sandwich ELISA, an expensive technique requiring bulky optical equipment and also labeling of the proteins. The long incubation times required (several hours) make this a rather time consuming technique. Proteins are also separated and recognized using Western blotting, based on gel electrophoresis, also requiring labeling of the proteins, making it an expensive and time consuming technique due to the reagent preparation required and the long separation time. Western blotting is typically used in conjunction with mass spectrometry to recognize and analyze proteins.
Protein microarrays have also provided the ability for multiplexed quantification and detection. However, similar to all other fluorescence based detection techniques, protein microarrays require long incubation times and high reagent costs. The use of impedance based sensors for the detection of biomolecules eliminate the need for fluorescent labeling and also provide the opportunity for multiplexed analysis of biomolecules due to the ease of integrating CMOS (complementary metal oxide semiconductor), thus being a good candidate for the clinical setting.
Nanogap sensors have been used to demonstrate detection of proteins. Using techniques such as resistive pulse sensing, changes in the size of functionalized microspheres have been used for demonstrating multiplexed target protein biomarker detection at concentrations as low as 15 ng ml-1. With further optimization, it may be possible to decrease the detection limit by one or two orders of magnitude to achieve the detection limits required for cancer detection (4 ng ml-1 for PSA). Capacitive electrical biosensors, based on changes induced by target molecule and probe binding on the surface charge on the electrode-electrolyte interface, have also been reported for detection of DNA hybridization and protein biomarkers. However, consistency in the results is problematic for these types of sensors. Recently, the electrical detection of protein biomarkers at detection limits as low as 1 pg ml-1 has also been demonstrated using nanowires, however the sensor operates at very low salt concentrations, making it incompatible with physiological conditions in clinical samples like blood. Nanowires are also more difficult and expensive to fabricate making it an unsuitable candidate for the clinical setting in the near future. Detection of protein biomarkers and nucleic acid biomarkers has been demonstrated at the single molecule level using solid-state nanopores.
A rapid and inexpensive methodology for detecting the hybridization of two DNA strands can be useful in detecting the presence of certain genes in a patient's DNA. By detecting such gene sequences it is possible to determine whether a patient has predisposition to a certain type of disease allowing him to get treatment to prevent the disease. Currently DNA hybridization is detected using techniques such the use of DNA microarrays and also real-time PCR. Such techniques are expensive given that they require the use of fluorescent labels which result in high reagent costs. The other major cost comes from the use of expensive and bulky optical scanners required for reading the fluorescent signals. DNA hybridization also requires overnight incubation given that thousands of molecules must hybridize in order to produce enough optical signal to be readable by the fluorescent scanner.
The main challenge for rapid detection of a single bacterial cell lies in establishing a procedure which is ultrasensitive and can detect in real time, while, at the same time, being inexpensive and easy to use. Recently, many efforts have been made toward the use of impedance based sensors for detection of bacterial cells. Impedance based sensors are advantageous since they eliminate the need for fluorescence labeling. Several groups have reported pathogen detection using electrical impedance sensors. Many of the electrical impedance sensors presented (with some exceptions) to date require numerous washing steps and lack the ability for real-time detection. As for detection time, flow-cytometry based methods such as the use of coulter counters, have provided the ability to analyze the dielectric properties of a cell in real time. With the on-chip integration of microchannels and microelectrodes, the ability to count, sort, and trap cells and analyze their dielectric properties has been demonstrated. This type of device would operate on the principle of measuring the current change caused by the displacement in the fluid as the particle passes by two measuring electrodes. A device relying solely on this principle has difficulty in differentiating between two different types of cells which may have similar dielectric properties. Thus, this type of device would have difficulty in detecting a target cell in a complex mixture. The use of electroosmotic trapping when used in conjunction with impedance spectroscopy is a promising method for detection of targeted particles.